Chronic viral infections remain major threats to global health, with pathogens such as hepatitis B virus (HBV) responsible for millions of deaths annually. Despite availability of an HBV vaccine and suppressive antiviral treatment, the worldwide burden of chronic HBV infection has remained unchanged. Short-term therapies capable of producing at least functional cure for chronic HBV are therefore a high priority. Exhaustion of the immune system, most obvious in HBV-specific CD4 and CD8 T cells, is a key factor in HBV persistence and disease. The PD-1:PD-L1/2 inhibitory receptor pathway regulates many key aspects of cellular immunity, including T cell exhaustion in chronic viral infection and cancer. Blockade of this pathway has produced dramatic effects in the treatment of advanced cancer and unleashed an immunotherapeutic revolution. However, little is known about the effect of PD-1 blockade in chronic infections in humans, the mechanisms by which responses are invigorated, and how a reinvigorated HBV immune response impacts the intrahepatic HBV replication landscape. We propose to comprehensively investigate how blockade of PD-1 in humans affects the layers of molecular regulation of the antiviral immune response, and how this impacts viral replication in situ. The overall hypothesis of this project is that blocking PD-1 in humans will alter the magnitude, quality, regulation and composition of pre-existing antiviral CD4 and CD8 T cell responses, leading to more effective viral control, will modulate other aspects of cellular innate immunity, notably macrophages, and will lead to diminished viral replication in hepatocytes. We will test this hypothesis through the following aims. Specifically, in Aim 1 we will test whether and how PD-1 therapy invigorates exhausted virus-specific CD4 and CD8 T cell responses in the blood and liver. We will utilize liver fine needle aspirates and PBMC samples for a comprehensive and in-depth analysis of changes in the phenotype, function, clonal composition, and transcriptional state of HBV- specific CD4 and CD8 T cells. In Aim 2 we will test whether PD-1 therapy diminishes active HBV transcription in hepatocytes. Using an integrated platform of single-cell laser capture microdissection and droplet digital PCR on frozen liver tissues, we will quantify cccDNA and pre-genomic HBV RNA in hundreds of individual hepatocytes from participants before and at the completion of PD-1 blockade. Finally, in Aim 3 we will define the recovery of macrophages through PD-1 blockade in persons with chronic HBV infection. Here using FNA and PBMCs from patients with chronic HBV, we will assess whether ?PD-1 treatment shifts the composition of macrophage populations toward an antiviral phenotype and test whether the functional responsiveness of monocyte/macrophages correlates with ?PD-1 treatment outcome. Collectively, we expect these data to dramatically enhance our understanding of the mechanism of ?PD-1 mediated immune recovery that can be utilized not only for the design of pathogen-specific immunotherapy, but also to enable further improvements in the efficacy of immunotherapy in general.